The present invention relates generally to the enzyme-catalyzed production of acetaldehyde from ethanol. More particularly, the invention relates to a process for the in situ generation of acetaldehyde in ethanol-containing orange essence. In addition, the present invention relates to a method for stabilizing aqueous solutions of acetaldehyde.
The origin of the present invention resides in work directed to the generation of acetaldehyde in orange essence. Orange essence is an aqueous distillate, obtained during the initial stages of evaporative concentration of orange juice, which contains the more volatile components of orange flavor. Aqueous essence is readded to concentrated orange juice before freezing to restore the fresh fruit flavor, and also is widely utilized as a flavoring ingredient in other citrus-based products.
Acetaldehyde, the primary aldehyde in orange essence, is known to exert a positive influence on orange flavor. In addition, acetaldehyde is an important constituent of many other fruit essences and, hence, assumes an important role not only in citrus products but in the flavor industry as a whole. It is desirable, therefore, to investigate processes for producing acetaldehyde and, particularly, for producing natural acetaldehyde, i.e., acetaldehyde generated by a natural process from a naturally-derived source.
Orange essence is a prime candidate for such a process since ethanol constitutes up to 90% of the total volatile content of the essence yet makes no positive contribution per se to the flavor. The ratio of ethanol to acetaldehyde in orange essence is on the order of 100 to 1. Thus, natural in situ conversion of the ethanol in orange essence to acetaldehyde is a potentially feasible means to produce acetaldehyde-enriched essence, provided such conversion can be accomplished without adversely affecting or altering other desirably present constituents of the ethanol source material.
Natural oxidation of ethanol can be achieved in a number of ways. One method, such as that described in U.S. Pat. No. 3,642,581 issued Feb. 15, 1972, to Risley and Goodhue, utilizes micro-organisms to perform the oxidation, wherein the ethanol source (substrate) is added to and the product recovered from the culture medium. This method is unsuitable for use with a material of complex composition such as essence because of the potential alteration of constituents other than ethanol/acetaldehyde.
A second approach, as taught by Leavitt and Pennington (U.S. Pat. No. 3,344,047 issued Sept. 26, 1967), uses the total cell-free enzyme extract from a microorganism grown in a nutrient broth wherein the desired substrate is the sole carbon source. Products are recovered either by solvent extraction or by emulsifying the aqueous solution with an oil phase into which the product is continuously extracted (Leavitt U.S. Pat. No. 3,880,739 issued Apr. 29, 1975). The oil phase-product solution is recovered using a semi-permeable membrane. While this process is more selective than the former, it is undesirable for use with a material of complex composition such as essence, again because of the potential number of undesirable enzyme reactions which may occur.
The greatest selectivity in naturally oxidizing ethanol is achieved using a specific enzyme, in this case alcohol dehydrogenase (ADH), to catalyze the oxidation. While in vivo utilization of this enzyme has been practiced for centuries in the fermentative production of ethanol by yeast, the pure enzyme has found little use in industry because of associated problems with retention and recycling of its required cofactor, nicotinamide adenine dinucleotide (NAD+). Solution of the former problem recently has been approached in several ways. Weibel et al., "Enzyme Engineering", Vol. 2, p. 203 (Plenum Press, N.Y. 10973); Weibel, "Interdisciplinary Research On Enzyme Systems With Special Emphasis On Redox Reactions", Report No. NSF/RA 761624, p. 33 (Nat'l. Tech. Info. Serv., Washington, D.C. 1976), and Bright, Ibid., p. 10, have immobilized nicotinamide adenine dinucleotide on a soluble high-molecular weight dextrin, and entrapped this immobilized cofactor and the ADH enzyme using a semi-permeable membrane. While activity and stability of such soluble immobilized cofactors are encouraging, these materials are, at present, impractical because of availability and economic considerations. Davis (U.S. Pat. No. 3,915,799 issued Oct. 28, 1975), teaches cofactor retention using a considerable excess of enzyme over cofactor whereby the cofactor is retained as a bound complex with the enzyme which is, in turn, confined by a semi-permeable membrane. This approach has the disadvantage that at any given time a large proportion of enzyme is inactive due to partial cofactor loading. Cofactor loss, while retarded, is not prevented.
A third approach is to use a membrane which is sufficiently tight so as to retain both enzyme and cofactor while allowing permeation of both substrate and product (Chambers, et al., "Enzyme Engineering", Vol. 2, p. 195 (Plenum Press, N.Y. 1973). This approach is disadvantageous because membranes which are sufficiently tight to retain NAD+ also significantly retard diffusion of smaller molecules, thus minimizing contact between enzyme and substrate.
In the enzyme-catalyzed conversion of ethanol or ethanol-containing products to acetaldehyde, regeneration of the required co-factor, which is reduced in the oxidation of ethanol, is desired in order to provide a commercially feasible process. Regeneration of the cofactor has been accomplished using a second enzyme, such as lactate dehydrogenase, to oxidize the reduced cofactor (NADH) while reducing a second substrate (Mossbach, et al., "Enzyme Engineering", Vol. 2, p. 143 (Plenum Press, N.Y. 1973)). This necessitates separation of products resulting from the two enzyme reactions. A similar approach described by Fink, Enzyme Technology Digest, 5, 52 (1976), but requiring no second enzyme, is to add to the reaction mixture benzaldehyde which is reduced by ADH during ethanol oxidation, thus recycling NADH to NAD+. This again requires separation of products. A third approach is to ultimately oxidize the reduced cofactor with molecular oxygen either via a suitable intermediate proton acceptor such as a flavin, or directly, using an enzyme such as diaphorase (Chambers, supra; Jones, Enzyme Technology Digest, 5, 50 (1976)).
In addition to these difficulties in providing a practical, enzyme-catalyzed process for converting ethanol, particularly ethanol contained as a component in a complex mixture such as essence, to acetaldehyde, this equilibrium conversion is thermodynamically unfavorable for appreciable oxidation of ethanol. Indeed, equilibrium considerations favor the reverse reaction, i.e., conversion of the acetaldehyde to ethanol, even under theoretically unfavorable conditions, such as providing ethanol in considerable excess. The equilibrium can be shifted in favor of formation of acetaldehyde, however, by removal of the acetaldehyde as it is formed. Traditionally, this has been accomplished either by forming a carbonyl addition product from acetaldehyde (e.g., with sodium bisulfite) or by enzymatic conversion of the acetaldehyde to a different product via an irreversible or thermodynamically favorable equilibrium reaction (e.g., oxidation of acetaldehyde to acetic acid). The undesirability of proceeding in this manner is self-evident where acetaldehyde or an acetaldehyde-enriched product is the desideratum.